Photon signal processing for particle detection

ABSTRACT

A system is described wherein multiple lasers are used to irradiate particles in a flow cytometer&#39;s flow stream. In certain embodiments, a light source having a first laser configured for continuous irradiation of a flow stream and one or more second lasers configured for irradiation of the flow stream in discrete intervals where each discrete interval of irradiation by the second laser is triggered by irradiation of one or more particles in the flow stream with the first laser. Methods for modulating the laser irradiation and measuring light intensity are also described. Also described is the computations and systems required to operate the one or more second lasers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a continuation of U.S. patentapplication Ser. No. 16/908,638 filed Jun. 22, 2020 which is related toand claims the priority benefit of U.S. Provisional Patent ApplicationSer. No. 62/865,107, filed Jun. 21, 2019, the contents of each of whichis hereby incorporated by reference in its entirety into the presentdisclosure.

STATEMENT REGARDING GOVERNMENT FUNDING

None.

TECHNICAL FIELD

The present disclosure relates to flow cytometry, and in particular to asystem and a sensor used therein suitable for single photon detection.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Flow cytometry is ubiquitously used in the fields related to lifesciences such as genetics, immunology, molecular biology, andenvironmental science. In general terms, flow cytometer/cytometry refersto a systems/method used to i) detect, and once detected ii) measurephysical and chemical attributes of particles moving along with a sheathfluid across an interrogation window such that only one such particleappears at a time for interrogation. Typically a source of light is usedto shine light at various wavelengths onto such particles. Light that isincident on such particles is scattered, typically in a forward or sidescatter and detected by photodetectors positioned about the direction ofsuch scatters. Light scattered from the particles are considered asemissions. These photodetectors are typically photodiodes orphotomultiplier tubes. In both cases, detectors generate electrons whenexcited by photons of the emitted light from the particles. Typicallythe current from the excited electrons is measured and labeled as thephotocurrent. The photocurrent can be correlated to general populationdata of the particles, some information about heterogeneity of thepopulation. Common light sources includes lasers. Common lasers includeultraviolet (UV) having a wavelength of 355 nm to 360 nm, violet havinga wavelength of 405 nm to 407 nm, blue having a wavelength of 488 nm,red having a wavelength of 633 nm, yellow having a wavelength of 561 nm,and green having a wavelength of 532 nm. Blue laser is found to be themost common.

The current flow cytometers provide useful but limited information aboutpopulation of particles including cells. Use of this technique allowsscientist to make measurements on a large number of particles in arelatively short period of time. Towards this end, the current flowcytometer systems provide the basis for a robust statistical analyses,giving way quantitative analyses of features. However, the informationthat these system provide are limited to population-based analyses. Forexample, information about heterogeneity of the population can beobtained from current flow cytometers. However, specific informationabout particles/cells, e.g., receptor sites, are seriously limited.Advances in the field have not been able to address these issues.Advances include deploying multiple lasers, hydrodynamic and acousticfocusing of particles in the flow tube, high-speed flows, and flowcytometry imaging. However, none of these advances address the baselineissue: photocurrent provides information about a stream of photons andat most statistical information can be obtained with limitedsensitivity, insufficient for precise detailed analyses ofparticle/cellular features.

Therefore, there is an unmet need for a novel flow cytometer system thatcan provide heightened sensitivity allowing discrimination betweenparticle/cellular features.

SUMMARY

A signal shaping sub-system for use with a flow cytometry system isdisclosed. The photon-accounting system includes a signal shapingsub-system. The signal shaping sub-system includes a differentiator thatis configured to be coupled to one or more Geiger-mode photodiodesproviding a capacitively coupled signal from the photodiodes. The signalform the one or more Geiger-mode photodiodes includes a combination ofavalanche portions and recharge portions associated with receivingphotons overlaid on one-another. The differentiator is configured togenerate a differentiated output of the signal into correspondingzero-crossings each associated with one of the received photons. Thesignal shaping sub-system further includes a comparator that isconfigured to receive the differentiated signal and compare to athreshold to thereby generate a comparator output digital signalassociated with the crossing of the differentiated signal about thethreshold. signal shaping sub-system further includes a frontendsynchronization system adapted to receive and synchronize the comparatorgenerated digital signal to a clock to thereby generate synchronizedphoton data with the clock and associated with the asynchronizedphotodiode signal. The signal shaping sub-system also includes atimestamping system adapted to receive the synchronized data as a bitstream and generate a timestamp associated with each photon data.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a flow cytometry system and its data flowbetween a flow cytometer and a data processor and user interfaceworkstation (or also referred to herein as a processor system).

FIG. 2 is a schematic of the flow cytometer of FIG. 1.

FIG. 3 is detailed block diagram of optical system of a flow cytometer.

FIG. 4 is a high-level diagram showing the components of an exampledata-processing system for controlling measurement systems, measuringdata, analyzing data, or performing other functions of a flow cytometrysystem of FIG. 1.

FIG. 5a is a schematic of a pn silicon device adapted to generate anelectron-hole pair when receiving incident light.

FIG. 5b is a cross sectional area of the pn device showing the avalancheregion.

FIG. 5c is a graph of current vs. reverse bias voltage showing threemodes: photodiode mode, avalanche photodiode mode, and the Geiger modein which mode the silicon photomultipliers (SiPM) is preferablyoperating.

FIG. 6a is an SiPM array, according to the present disclosure.

FIG. 6b is a sample schematic showing how the Geiger avalanchephotodiode (APD) and quench resistors are connected to each other in thearray.

FIGS. 7a and 7b are graphs of current vs. bias and voltage vs. time,respectively, which show examples of the SiPM behavior during anavalanche and quenching process.

FIG. 8 is a graph of detector output vs. time, providing the raw datawhich shows two photon events on the double peak around 1.5 ns, and asingle photon peak near 3 ns.

FIG. 9a is a circuit diagram, for demonstration purposes, which showsuse of a differentiator to provide 1 ns photon pulses.

FIG. 9b is an example of a single photon events with an enlarged view ofthe differentiated signal as compared to the raw data at the moment ofthe avalanche event.

FIG. 9c is an example of a multiple photon events with an enlarged viewof the differentiated signal as compared to the raw data at the momentof the avalanche events during the same quenching period.

FIG. 10a is an example differentiation circuit with a high pass sectionand a differentiation section.

FIGS. 10b and 10c are example graphs showing results of the circuitshown in FIG. 10a , where in FIG. 10b an example input to thedifferentiation stage of the circuit is shown and FIG. 10c shows anexample output from that circuit.

FIG. 11a is an adaptive threshold circuit.

FIG. 11b is output of the circuit shown in FIG. 11 a.

FIG. 11c is a circuit of an example filter section in anenvelope-detector of that shown in FIG. 11 a.

FIG. 11d is an example output of the circuit shown in FIG. 11 c.

FIG. 12a is a circuit according to the present disclosure that can thenbe used to count pulse-widths and be used as a discriminator.

FIG. 12b is a timing diagram showing interim signals of the circuitshown in FIG. 12 a.

FIGS. 13a and 13b are example circuit configurations of adaptivepedestal clamping circuits.

FIGS. 14a and 14b , are values of pixel out vs. time for low photoncount rate and high photon count rate, respectively.

FIGS. 14c and 14d are values of differentiation out vs. time for lowphoton count rate and high photon count rate, respectively.

FIGS. 14e and 14f are values of adaptive pedestal clamp vs. time for lowphoton count rate and high photon count rate, respectively.

FIG. 15 is a block diagram of a signal shaping sub-system according tothe present disclosure.

FIGS. 16a and 16b are frontend digital circuits for synchronizing anotherwise asynchronous digital signal, according to the presentdisclosure.

FIG. 17 is a timing diagram is provided to show the timing relationshipof the frontend digital circuit of FIG. 16 a.

FIG. 18 is a block diagram on how the digital engine of the photoncounting operates, referred to herein as the photon pipeline.

FIG. 19 is an example discriminator circuit of the block shown in FIG.18, using a first discrimination criterion.

FIG. 20 is an example bitstream of another discrimination criterion.

FIG. 21 is a bitstream showing the operation of the priority encodershown in FIG. 18.

FIG. 22 is a series of bitstream showing exemplary operation of thepriority encoding and masking.

FIG. 23 is a circuit showing the capability to perform photon accountingat the same time as (simultaneously) as photocurrent detection.

FIG. 24 is a schematic of how each photon is timestamped in memory.

FIG. 25 is a Gaussian graph showing the output of application of amoving average filter to the timestamped photons of FIG. 24.

FIG. 26 is a schematic of a peak/width detection utilizing a cascade ofmoving average filters (1 . . . N stages).

FIG. 27 is a graphical representation of a system with multiple laserswhich provide an example of multiple laser excitation showing number ofpossible emission channels given a 42 channel detector array.

FIG. 28 is a schematic of a multi-laser system which provides an exampleof an optical distributor showing multiple excitation lasers, photodiodes and dichroic mirrors with light focused through an optical fiberto a flow chamber and emission light collected by an optical fiberconnected to a spectral detection unit.

FIG. 29 is a timing chart depicting the operational timing of the lasersshown in FIGS. 27 and 28 in response to a trigger signal from acontinuously irradiating laser according to certain embodimentsaccording to the present disclosure.

FIG. 30 is a block diagram of an optical system depicting function of anoptical modulator (AOM) that allows modulation of a laser beam.

FIG. 31 is a schematic of component integration for optical systems thatshows how pigtailed lasers can be combined and connected via standardconnectors and a system for laser modulation.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

In the present disclosure, the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range.

In the present disclosure, the term “substantially” can allow for adegree of variability in a value or range, for example, within 90%,within 95%, or within 99% of a stated value or of a stated limit of arange.

A novel flow cytometer system is disclosed that can provide heightenedsensitivity allowing discrimination between particle/cellular features.The novel system provides simultaneous capabilities to measurephotocurrent from photodetectors as well as detect and count photonswhich allows absolute quantification of particle and cellular features.Towards this end, the novel system receives photons at photosensorswhich excite corresponding electrons that are sensed, synchronizedagainst a clock, and time addressed for enhanced analysis using aGaussian photonic profile thereby generating pseudo-photocurrentwaveform from individually detected photons. Simultaneously, the systemof the present disclosure is capable of measuring photocurrent and thusprovide a direct photocurrent results. In addition, the presentdisclosure describes a novel photosensor capable of sensing photonsthereby generating an electronic signal associated with each photon,with selective passive-active quenching, synchronizing an otherwiseasynchronous electronic signal, and placing the synchronized signal intohigh-speed memory utilizing direct memory access for superior overallresponse time generating extremely high sensitivity.

Referring to FIG. 1, a block diagram of a flow cytometry system 100 andits data flow between a flow cytometer 102 and a data processor and userinterface workstation 104 (or also referred to herein as a processorsystem 104). Within the flow cytometer 102 is a sensor system 106responsible for detecting particles. Particle data is transferred fromthe flow cytometer 102 to the processor system 104. The processor system104 in turn controls hardware of the flow cytometer 102 and there aresignals from the flow cytometer 102 controlling user interface of theprocessor system 104. From a high level, the flow cytometry system 100is capable of providing individual photon information, e.g., time ofarrival; a pseudo-Gaussian photon distribution from the photon data, anda simultaneous photocurrent from the received light upon thephotosensors.

An example of the flow cytometer 102 is shown in FIG. 2. Referring toFIG. 2, a schematic of a flow cytometer 200 is provided. The flowcytometer 200 can be used with sensors and detection systems describedherein. The flow cytometer 200 includes a flow cell 202. The flow cell202 includes a flow chamber 204, and is, at least in part, transparentor substantially transparent to irradiation such as incident light Lfrom a light source 208. Resultant light also referred to herein asemission light are emitted from the flow chamber 204 when a particle,e.g., a cell, passes through the chamber. The emission light are basedon forward and side scatter, such as light L_T FS (forward scatter) andL_T SS (side scatter). Without a particle in the flow chamber 204, thelight is substantially without scatter. For clarity, only part of theflow chamber 204 is shown. Further details of various configurations ofthe flow chamber 204 are discussed below. As shown, the flow cell 202can be 2 mm thick along the direction of propagation of light L,however, other dimensions are within the ambit of the presentdisclosure. The narrowness of the flow chamber 204 within the flow cell202 is related to hydrodynamic focusing, which is a concept of narrowingthe flow chamber 204 so that only one particle can pass at a time. Assuch, Hydrodynamic focusing is actually the narrowing of the core stream(containing particles) so that the velocity increases, separatingparticles so that a single particle can be analyzed in the absence ofanother. As the sheath fluid compresses the core, the velocity increasesand the particle remains in the center of the stream to enhancestability of particle location. However, if the particles are notcentered improper scattering results. Therefore, hydrodynamic focusingalone may not be sufficient. To further improve centering of particlesin the flow chamber 204, acoustic focusing, known to a person havingordinary skill in the art may also be implemented to center particleswithin the flow chamber 204 for proper positioning.

A sheath flow SH flows into the flow cell 202 from an inlet port IN1.For example, saline, which is an isotonic liquid, or water, can be usedas the sheath flow SH. However, the sheath flow SH is not limited tosaline, but various types of liquid such as water, other aqueoussolutions (whether isotonic or not), and organic solvents can be used.In various examples, the sheath flow SH also flows into the flow cell202 from at least one additional inlet port, shown as IN3. Multipleports allow the sheath to “escape” (to thereby balance input-output) asthe amount that can leave the orifice is very small even though thestream is moving much faster.

Further, a sample flow SM including microparticulate samples or othertargets to be analyzed flows into the flow cell 202 from an inlet portIN2. For example, saline, which is an isotonic liquid, can be used asthe sample flow SM. However, the sample flow SM is not limited tosaline, but various types of liquid such as water, other aqueoussolutions (whether isotonic or not), and organic solvents can be used.The inflow pressure of the sample flow SM can be higher than or lowerthan the inflow pressure of the sheath flow SH. The flow chamber 204 orother fluid channels in the flow cell 202 can be arranged so that thecenter of the sample flow is fastest and the flow velocity is near zeroat the walls of the flow channel 204. This can cause targets to behydrodynamically focused, i.e., positioned by the sheath fluid flow,substantially in the center of the sample flow. In the illustratedexample, the fluid flows SM and SH are provided by a fluidic supply 206.It should be noted that sample may flow in the upward or downwarddirections depending on the instrument design (analyzers typically flowup to prevent air bubbles causing turbulence and sorter always flowdown).

The inlet ports IN1, IN2, IN3 can be bored, molded, or otherwise formedin the flow cell 202. In an example, the flow cell 202 includes glass orquartz. For example, flow channels (e.g., flow chamber 204) can beformed by micro-blasting of quartz sheets. Ports IN1, IN2, IN3 can bedrilled out of the quartz sheets. Other etching and boring techniquescan be used to form flow channels, inlets, and other features. Forexample, sample channels, including the flow chamber 204, can be etched,and sheath channels can be micro-blasted using a mask to define thedesired pattern. In other examples, channels and other cavitiesdescribed herein can be injection molded, molded using other techniques,bored, or etched.

The sheath flow SH and the sample flow SM merge in the flow chamber 204,so that a flow F is provided in which the sample flow SM issubstantially hydrodynamically focused with the sheath flow SH, e.g.,around the sample flow SM, or arranged in other hydrodynamic-focusingconfigurations. The flow F can be discharged to the outside of the flowcell 202 in some examples. The flow F can move at a predetermined flowrate. The flow rate can be expressed as volumetric flow rate (e.g., inμL/s or mL/min), or as linear flow rate of a sample (e.g., amicroparticle) hydrodynamically focused within, and moving with, thesample flow (e.g., in m/s).

The optical source 208, e.g., a laser or other illumination source, canprovide light L aimed, focused, or otherwise directed to irradiate thetargets entrained within the sample flow SM. The laser light L incidenton a particle can be at least partly transmitted or at least partlyscattered, providing resultant forward-scattered light L_T FS andresultant side-scattered light included in L_F SS. Targets, e.g.,chromophores bound to target molecules of interest, can fluoresce,producing resultant fluorescent light also included in L_F SS. Adetector 210, e.g., an on-axis detector, can detect light L_T FS. Adetector 212, e.g., a perpendicular detector, can detect light L_F SS.Various embodiments can use one detector or more than one detector.Detectors can be placed at any angle with respect to the axis of thelight L.

According to various embodiments, the light source 208 can be varioustypes of lasers. For example, the light source can be one or more ofultraviolet (UV) having a wavelength of 355 nm to 360 nm (also 325 nmand 349 nm are used), violet having a wavelength of 405 nm to 407 nm,blue having a wavelength of 488 nm, green having a wavelength of 532 nm,yellow having a wavelength of 561 nm, and red having a wavelength of 633nm or near IR at 808 nm. In addition, according to one embodiment, theoptical source 208 includes a laser and conjugate focusing optics, anddetector 210 includes a motorized monochromator, one or more siliconphotomultipliers (SiPM), and a Geiger-mode differential detectioncircuit, further described below.

In some examples, a controller (“CTL”) 214 controls operation of thefluidic supply 206 or the optical source 208. In some examples,controller 214 receives information, e.g., photon counts, from detectors210, 212. In other examples, the controller 214 is placed in theprocessor system 104 (see FIG. 1).

Referring to FIG. 3, a more detailed block diagram of optical system 300of a flow cytometer is shown. FIG. 3 represents both a top view as wellas a sideview. As discussed above, a light source and the associatedoptical elements, e.g., lenses, 302 provide incident light on to a flowchamber 304 which then causes forward and side scatter when the lightstrikes a particle, e.g., a cell in the flow chamber 304. Variousdetectors and their associated optics 306 and 308 are shown as detectorsand optical elements for forward and side scatter, respectively.

Referring to FIG. 4, a more detailed block diagram of the block diagramshown in FIG. 1 is provided. FIG. 4 is a high-level diagram 400 showingthe components of an example data-processing system 401 (which mayrepresent processor system 104 of FIG. 1) for controlling measurementsystems, measuring data, analyzing data, or performing other functionsdescribed herein, and related components. The system 401 includes aprocessor 486, a peripheral system 420, a user interface system 430, anda data storage system 440. The peripheral system 420, the user interfacesystem 430, and the data storage system 440 are communicativelyconnected to the processor 486. Processor 486 can be communicativelyconnected to network 450 (shown in phantom), e.g., the Internet or aleased line, as discussed below. Devices above can each be or includeone or more of systems 401, 486, 420, 430, or 440, and can each connectto one or more network(s) 450. Processor 486, and other processingdevices described herein, can each include one or more microprocessors,microcontrollers, field-programmable gate arrays (FPGAs),application-specific integrated circuits (ASICs), programmable logicdevices (PLDs), programmable logic arrays (PLAs), programmable arraylogic devices (PALs), or digital signal processors (DSPs).

Processor 486 can implement processes of various aspects describedherein. Processor 486 and related components can, e.g., carry outprocesses for detecting photons, collecting count data from counters,operating a laser (e.g., adjusting the laser power), operating amonochromator (e.g., to scan across a wavelength band), or operating afluid supply or other components of a flow cytometer 102 as in FIG. 1.

Processor 486 can be or include one or more device(s) for automaticallyoperating on data, e.g., a central processing unit (CPU),microcontroller (MCU), desktop computer, laptop computer, mainframecomputer, personal digital assistant, digital camera, cellular phone,smartphone, or any other device for processing data, managing data, orhandling data, whether implemented with electrical, magnetic, optical,biological components, or otherwise.

The phrase “communicatively connected” includes any type of connection,wired or wireless, for communicating data between devices or processors.These devices or processors can be located in physical proximity or not.For example, subsystems such as peripheral system 420, user interfacesystem 430, and data storage system 440 are shown separately from theprocessor 486 but can be stored completely or partially within theprocessor 486.

The peripheral system 420 can include or be communicatively connectedwith one or more devices configured or otherwise adapted to providedigital content records to the processor 486 or to take action inresponse to processor 486. For example, the peripheral system 420 caninclude digital still cameras, digital video cameras, cellular phones,or other data processors. The processor 486, upon receipt of digitalcontent records from a device in the peripheral system 420, can storesuch digital content records in the data storage system 440. In theillustrated example, the peripheral system 420 permits the processor 486to control fluidic supply 206 (see FIG. 2) and optical source 208 (seeFIG. 2).

The peripheral system 420 also permits the processor 486 to receive datafrom detector(s) 402. Detectors 402 can include optical detectors, splitphotodiode; split or position-sensitive photodiode; photon sensor,photodiode(s), single or arrayed Si avalanche photodiode(s) (SiPMs).Additionally or alternatively, the detectors 402 may include customizedintegrated circuits which include various elements such as activequenching, analog-to-digital converters, digital synchronizationcircuits, and onboard memory for extreme fast logging of photon data, asdescribed below. Additionally or alternatively, detectors 402 caninclude electrical detectors, including a photocurrent detectorincluding dQ/dt, LPF/integrator, upper ADC, photon charge detectionblocks including integrating amplifier, lower ADC, Normal out, Low-passfilter, or photocurrent detection blocks, including ADC.

In some examples, the peripheral system 420 also permits the processor486 to control a spectral discriminator 460, e.g., a motor or otherdrive that operates a monochromator to select the particular wavelengthband output by the monochromator.

In some examples, the peripheral system 420 permits the processor 486 tocontrol a drive circuit 462. Drive circuit 462 can provide a biasvoltage or other drive voltages, e.g., to operate detectors 402.Processor 486 and drive circuit 462 can control bias voltage in anopen-loop manner, or in a closed-loop manner, e.g., using any of, orcombination of any of, proportional (P), integral (I), and derivative(D) control, or other closed-loop control techniques. Bias voltage canbe adjusted to maintain sensor readings within a range of the detectors402.

Detectors 402 or peripheral system 420 may include one or moreanalog-to-digital converters (ADCs) or digital-to-analog converters(DACs). An ADC or DAC is limited in the analog signals it can input oroutput, respectively, by: the supply rails powering it, which sets therange it can accept or produce (signals will clip at the limits of therange); its bit depth, which sets the granularity of signals within thatrange it can detect or produce; and its speed, which determines howquickly it can provide a sample. Some ADCs/DACs (e.g.,successive-approximation) permit trading off speed against bit depth(reduced speed for greater bit depth, or vice versa). In addition, someADCs/DACs saturate and become nonlinear when accepting or producingsignals within the supply rails but close to those rails (e.g., within0.7 V of the rails). Passives and other components used in associationwith ADCs/DACs can also affect ADC/DAC performance.

Similar limitations apply to other electronic measurement devices andsystems. For example, FPGAs configured to detect pulses have a maximumfrequency of operation of their input latches or deserializers. Ifpulses arrive faster than that maximum frequency, not all pulses will becounted. In some examples herein, a measurement device (e.g., an ADC, oran ADC and related components) is associated with a predetermined rangeof levels (e.g., for a 5V ADC, 0.7 V-4.3 V; for a counter, 1 Gsps).

Accordingly, in some examples, the processor 486 operates drive circuit462 to maintain analog signal levels at ADC inputs, counts at FPGA-basedor other counters, or other outputs from photodetectors described herein(e.g., photodiode arrays, and similar) within the predetermined ranges.A processor, and ASIC, or an FPGA can measure the rate at which photonsare being counted, and can analytically determine the derivative (ordifferential, finite difference, or other discrete approximation to thederivative) of the rate. If the rate exceeds a predetermined percentageof measured peak rate, or if the derivative indicates that the rate willsoon exceed the predetermined percentage, the processor 486 can reducethe bias to reduce the number of photon pulses (e.g., by reducing thenumber of pulses that exceed a comparator threshold). Therefore, afeedback control system can be generated.

Still referring to Paper 10, p. 25, in some examples, the processor 486can use the derivative to determine when to start and stop capturingdata. For example, when a derivative that has been increasing reaches amaximum and begins to decrease, the processor can determine that aGaussian particle-signal profile is at −1 standard deviation (σ), andcan begin capturing. Other derivatives, e.g., higher-order derivatives,or zero-crossings of functions or derivatives, can be used to indicatetiming for data capture. This can provide more consistency between thedata captured for each particle.

In some examples, the peripheral system 420 permits the processor 486 tocontrol a positioner 464, which can represent an x y shifter. Processor486 can operate positioner 464 in an open-loop manner, or in aclosed-loop manner, e.g., using any of, or combination of any of,proportional (P), integral (I), and derivative (D) control, or otherclosed-loop control techniques. Processor 486 can operate positioner 464to maintain alignment of one or more illumination spots in a sensingvolume. This can permit more accurately enabling individual spots(lasers) to measure particles flowing through a flow chamber.

The user interface system 430 can convey information in eitherdirection, or in both directions, between a user 438 and the processor486 or other components of system 401. The user interface system 430 caninclude a mouse, a keyboard, another computer (connected, e.g., via anetwork or a null-modem cable), or any device or combination of devicesfrom which data is input to the processor 486. The user interface system430 also can include a display device, a processor-accessible memory, orany device or combination of devices to which data is output by theprocessor 486. The user interface system 430 and the data storage system440 can share a processor-accessible memory.

In various aspects, processor 486 includes or is connected tocommunication interface 415 that is coupled via network link 416 (shownin phantom) to network 450. For example, communication interface 415 caninclude an integrated services digital network (ISDN) terminal adapteror a modem to communicate data via a telephone line; a network interfaceto communicate data via a local-area network (LAN), e.g., an EthernetLAN, or wide-area network (WAN); or a radio to communicate data via awireless link, e.g., WIFI or GSM. Communication interface 415 sends andreceives electrical, electromagnetic, or optical signals that carrydigital or analog data streams representing various types of informationacross network link 416 to network 450. Network link 416 can beconnected to network 450 via a switch, gateway, hub, router, or othernetworking device.

In various aspects, system 401 can communicate, e.g., via network 450,with a data processing system 404, which can include the same types ofcomponents as system 401 but is not required to be identical thereto.Systems 401 and 404 can be communicatively connected via the network450. Each system 401, 404 can execute computer program instructions tooperate measurement systems or capture measurements as described herein,or to communicate measurement data, e.g., via network 450.

Processor 486 can send messages and receive data, including programcode, through network 450, network link 416, and communication interface415. For example, a server can store requested code for an applicationprogram (e.g., a JAVA applet) on a tangible non-volatilecomputer-readable storage medium to which it is connected. The servercan retrieve the code from the medium and transmit it through network450 to communication interface 415. The received code can be executed byprocessor 486 as it is received, or stored in data storage system 440for later execution.

Data storage system 440 can include or be communicatively connected withone or more processor-accessible memories configured or otherwiseadapted to store information. The memories can be, e.g., within achassis or as parts of a distributed system. The phrase“processor-accessible memory” is intended to include any data storagedevice to or from which processor 486 can transfer data (usingappropriate components of peripheral system 420), whether volatile ornonvolatile; removable or fixed; electronic, magnetic, optical,chemical, mechanical, or otherwise. Example processor-accessiblememories include but are not limited to registers, floppy disks, harddisks, tapes, bar codes, Compact Discs, DVDs, read-only memories (ROM),erasable programmable read-only memories (EPROM, EEPROM, or Flash), andrandom-access memories (RAMs). One of the processor-accessible memoriesin the data storage system 440 can be a tangible non-transitorycomputer-readable storage medium, i.e., a non-transitory device orarticle of manufacture that participates in storing instructions thatcan be provided to processor 486 for execution.

In an example, data storage system 440 includes code memory 441, e.g., aRAM, and disk 443, e.g., a tangible computer-readable rotational storagedevice or medium such as a hard drive. Computer program instructions areread into code memory 441 from disk 443. Processor 486 then executes oneor more sequences of the computer program instructions loaded into codememory 441, as a result performing process steps described herein. Inthis way, processor 486 carries out a computer implemented process. Forexample, steps of methods described herein, blocks of the flowchartillustrations or block diagrams herein, and combinations of those, canbe implemented by computer program instructions. Code memory 441 canalso store data, or can store only code.

Furthermore, various aspects herein may be embodied as computer programproducts including computer readable program code (“program code”)stored on a computer readable medium, e.g., a tangible non-transitorycomputer storage medium or a communication medium. A computer storagemedium can include tangible storage units such as volatile memory,nonvolatile memory, or other persistent or auxiliary computer storagemedia, removable and non-removable computer storage media implemented inany method or technology for storage of information such ascomputer-readable instructions, data structures, program modules, orother data. A computer storage medium can be manufactured as isconventional for such articles, e.g., by pressing a CD-ROM orelectronically writing data into a Flash memory. In contrast to computerstorage media, communication media may embody computer-readableinstructions, data structures, program modules, or other data in amodulated data signal, such as a carrier wave or other transmissionmechanism. As defined herein, computer storage media do not includecommunication media. That is, computer storage media do not includecommunications media consisting solely of a modulated data signal, acarrier wave, or a propagated signal, per se.

The program code includes computer program instructions that can beloaded into processor 486 (and possibly also other processors), andthat, when loaded into processor 486, cause functions, acts, oroperational steps of various aspects herein to be performed by processor486 (or other processor). Computer program code for carrying outoperations for various aspects described herein may be written in anycombination of one or more programming language(s), and can be loadedfrom disk 443 into code memory 441 for execution. The program code mayexecute, e.g., entirely on processor 486, partly on processor 486 andpartly on a remote computer connected to network 450, or entirely on theremote computer.

The full block diagram for the components used in various embodiments isshown in FIG. 15, described further below. Each of these components isfirst described with reference to FIGS. 5a-5c, 6a-6b, 7a-7b , 8, 9 a-9c, 10 a-10 c, 11 a-11 b, 12 a-12 b, 13 a-13 b, 14 a-14 f, and 15 a-15 b,each of which is described in detail below.

As discussed above, several types of optical detectors can be used withthe systems described in the present disclosure. These includephotodiodes, photomultiplier tubes, SiPM, Geiger-mode photodiodes, andother types of light sensitive sensors known to a person having ordinaryskill in the art. Referring to FIGS. 5a-5c operations of an example SiPMis described. FIG. 5a is a schematic of a pn silicon device adapted togenerate an electron-hole pair when receiving incident light. The pndevice is coupled to a voltage source for biasing and a resistor R. Inthe Geiger region of operation, the bias voltage is sufficiently high inmagnitude that, when a photon strikes the sensor and releases aphotoelectron, that electron can strike other atoms and releaseadditional electrons. Accordingly, a single photon can trigger a cascadeof electrons that causes the SiPM to become conductive and produce adetectable current sensed at R. FIG. 5b shows a cross sectional area ofthe pn device showing the avalanche region. Thus when incident photonstrikes the pn device, electrons are generated according to the Geigermode which is extremely fast on the order of 10 ps. FIG. 5c is a graphof current vs. reverse bias voltage showing three modes: photodiodemode, avalanche photodiode mode, and the Geiger mode in which mode theSiPM is preferably operating.

FIG. 6a shows example configurations of an SiPM array 600. As shown, asensor can include rows and columns of sensor elements (i.e., pndevices, see FIG. 5b ). Each sensor element can include a quenchresistor in series with a Geiger-mode avalanche photodiode (APD), e.g.,a SiPM as shown in FIG. 5b . The sensor elements can be electrically inparallel across a row, column, or 2 D sensor array. As current flowsthrough the APD in response to impact of a photon thereon, voltageacross the quench resistor increases. Therefore, voltage across the APDdecreases. When the voltage across the APD drops below the Geigerthreshold because of the quenching resistor, the APD will cease toconduct and will be ready to detect another photon. This process isreferred to as “quenching.” FIG. 6b is a sample schematic showing howthe Geiger APD and quench resistors are connected to each other in thearray 600. Each Geiger-mode avalanche photodiode is coupled to a quenchresistor with a differential output to be read with downstreamcircuitry. The common terminals of the Geiger APDs is coupled to abiasing circuit that biases the diodes above the breakdown voltage ofeach diode. The common terminal of the quench resistors is resistivelycoupled to ground providing a single-ended output that can also be readas a reference, as will be described in further detail below. Threeoptional outputs are shown: Vout_Photocurrent, Vout_PhHP, and Vout_Ph.The Vout_Photocurrent provides the capability to simultaneously measurethe photocurrent as well as the individual photons. Vout_PhHP is theoutput according to one style of the array in which the output of theAPDs pass through a high-pass filter prior to being further processed.Vout_Ph is the output of the array without high-pass filtering.

An arrayed pixel Si Photomultiplier (Geiger mode) such as thatillustrated in FIG. 6a can provide a high gain (e.g., >10⁶) and highsensitivity of photon detection. Such sensors can be relatively compactand can operate with relatively low bias voltages, e.g., <25-70 V. Suchsensors can be durable under light exposure and can be relativelyinsensitive to magnetic fields. However, some prior schemes have alimited dynamic range due to the limited number of pixels on thedetectors and the dead time during quenching, during which those priorsensors do not detect photons. Moreover, some prior sensors haverelatively high dark count rates (i.e., appreciable output signal evenwhen no light is incident) or can be sensitive to temperaturevariations.

FIGS. 7a and 7b which are graphs of current vs. bias and voltage vs.time, respectively, show examples of the SiPM behavior during anavalanche and quenching process. As discussed above, the avalancheprocess is very fast, e.g., on the order of picoseconds (e.g., 10 ps-100ps). However, the quenching (“recharge” or “dead time”) process iscomparatively slow, e.g., on the order of nanoseconds (e.g., 50 ns-100ns). As noted above, during some prior schemes, the sensor does notdetect photons during the recharge process. Accordingly, some priorsensors only provide a dynamic range of about three orders of magnitude.This inability poses a serious challenge when designing a system thatcan simultaneously measure photocurrent as well as single-photondetection, as there may be multiple photons incident during one fullcycle (i.e., avalanche-recharge/quenching). Referring to FIG. 8 which isa graph of detector output vs. time, the raw signal is shown which showstwo photon events on the double peak around 1.5 ns, while a singlephoton peak near 3 ns. The challenge with the first double photon eventis that the second photon arrived prior to the completion of thequenching phase of the first photon. As a result, there is uncertaintyas to how to measure the waveform.

One approach to overcome this challenge, according to the presentdisclosure, is to differentiate the raw signal. Referring to FIG. 9a ,for demonstration purposes, a circuit diagram is shown using adifferentiator to provide 1 ns photon pulses. This circuit can providedetection of multiple photons using a single pixel, which can in turnincrease dynamic range. The output of the detector is provided to thedifferentiator thereby causing a differentiation of the detected signal.Referring to FIGS. 9b and 9c two examples of the differentiation isshown. FIG. 9b provides an example of a single photon events with anenlarged view of the differentiated signal as compared to the raw dataat the moment of the avalanche event. Conversely, FIG. 9c provides anexample of a multiple photon events with an enlarged view of thedifferentiated signal as compared to the raw data at the moment of theavalanche events during the same quenching period. As shown in bothFIGS. 9b and 9c , the differentiated signals show pulses even when aphoton strikes the detector during the dead time (i.e., during thequenching period). Although the differentiator is shown as being coupledto the anode side of the APDs, it can additionally or alternatively becoupled to the cathode side of the APDs.

The number of photons is dependent on the power of the light source 208(see FIG. 2). A positive correlation between power and photons exists.

Signal processing of the differentiation signal can distinguish photonswithin overlapped photon pulses. Consequently, a more accurate photoncounting can be achieved owing to counting of photons that wouldotherwise be missed during an APD's dead time. Additionally, a fastdetection allows rapid measurements of the lifetime of fluorescentmolecules, something that is not possible with current state of the artflow cytometers.

Referring to FIG. 10a , according to various embodiments, an exampledifferentiation circuit is shown with a high pass section and adifferentiation section. The values of the components illustrated inFIG. 10a can be adjusted, e.g., to provide differentiation signalprocessing with a rise time T_(rise) corresponding to the timescale ofthe avalanche process with respect to a particular SiPM, e.g., 10 ps-100ps. In the illustrated circuit of FIG. 10a , an ideal square pulseproduces a Gaussian pulse after the high pass filter. Circuit parametersof the differentiation section can be adjusted to provide separation oftwo partially-overlapping Gaussian pulses. According to variousembodiments, example circuit parameters include:

High-pass filter: C_(HP) (Farad) = T_(RISE)/50 Differentiation filter:C_(DIFF) (Farad) = (1/5)CHP = T_(RISE)/250 Primary corner frequency: F0(Hz) = 1/(10 T_(RISE)) T_(RISE): avalanche process time; typical <100 ps

In FIG. 10a , there is also a transimpedance amplifier circuit to beused by a pedestal control circuit discussed below. The output of thetransimpedance amplifier is a voltage representation of the currentprovided by the APD. When the signal is passed through the high passfilter, because of AC coupling, the signal attains a pedestal. Thishappens specially when there are large number of photons affecting pulsewidths and frequency of photon events. Thereafter a pedestal controlcircuit is used to remove the deviation caused by the pedestal. Theoutput of the transimpedance amplifier is an enablement for the pedestalcontrol circuit, as discussed below.

Referring to FIGS. 10b and 10c , example results of the circuit shown inFIG. 10a are shown. In particular, FIG. 10b shows an example input tothe differentiation stage of the circuit and FIG. 10c shows an exampleoutput from that circuit. As shown, the differentiation stage respondsto two overlapping pulses, e.g., triggered by two separate photons, byproviding a signal having two zero-crossings (FIG. 10c ). Otherthresholds than zero can additionally or alternatively be used. Asindicated by the double-ended arrows in the top plot, a multiple-photonpulse can have a wider pulse width (longer duration) than asingle-photon pulse. Accordingly, a pulse width differential can be usedto discriminate closely overlapped 1-photon pulses from 2 photon pulses.One or more comparator/counter circuits can be used to sense the zerocrossings and count pulses.

The waveform shown in FIG. 10c can be prone to AC-coupling of outputsfrom the differentiator section, thereby large excursions of the signalbaseline can occur among rapid successive pulses. To reduce miscounts,according to various embodiments, an adaptive threshold level(s) isneeded by tracking the signal baseline. An RF envelope detector, e.g.,monolithic or made from discrete components, can produce a signal thatclosely follows peak excursions of the input. A filter can then furthersmooth the signal. A constant offset can also be added to the envelopeto produce the tracking threshold level(s). The pulse is delayed, e.g.,through at least one of a long transmission line or a passive network,so that the pulse arrives to the comparator at the same time as themoving threshold(s). Such an adaptive threshold circuit is shown in FIG.11a , with its output shown in FIG. 11b . Two thresholds areillustrated: a large-pulse threshold and a small-pulse threshold. Insome examples, the small-pulse threshold is used to detect single-photonexcitation (1PE) pulses and the large-pulse threshold is used to detectdouble-photon excitation (2PE) pulses. A 2PE pulse can result fromnear-simultaneous absorption of two photons, or from absorption of asingle photon of a shorter wavelength.

Referring to FIG. 11c , a circuit of an example filter section in anenvelope-detector circuit is shown. Output of the circuit of FIG. 11c isshown in FIG. 11d . The illustrated filter section can be used, e.g., inthe adaptive threshold circuit of FIG. 11a . In some example envelopedetectors, the diode is specified for GHz operation with junctioncapacitance, e.g., about 1 pF-about 10 pF. Some example envelopedetectors include a transimpedance amplifier (TIA).

Having developed the adaptive thresholds, the circuit shown in FIG. 12acan then be used to count pulse-widths and be used as a discriminator,according to various embodiments. This circuit is alternatively used ascompared to the photon pipeline discussed later. It should beappreciated that a photon even requires timing resolution in the psrange, therefore, comparators and counters capable of that kind ofresolution while may be available are excessively costly. Therefore, theimplementation shown in FIG. 12a is provided as an alternativeembodiment only.

With reference to FIG. 12a , there are two comparators: Comp1 and Comp2.In this implementation, Comp1 is left enabled, while output of Comp1enables Comp2 after a delay to. Input from the differentiator circuit(see, e.g., FIG. 10a ) is provided to Comp1 using an adaptive thresholdprovided by the circuit of FIG. 11a . The output of Comp1 is the fedboth to a lower counter and also used as an enable signal for Comp2. Thedifferentiator output is delayed by a delay that is less than to andpassed to Comp2. The output of Comp2 is then passed on to an uppercounter. As a result, if a pulse-width output of the differentiator issufficiently long corresponding to a double-photon event, Comp2 isenabled during the latter part of the pulse-width. Conversely, if thepulse-width output of the differentiator is short corresponding to asingle-photon event, Comp2 is enabled after the pulse has already ended.If effect, Comp2 stretches the pulse so that the counters can accuratelycount the pulses. FIG. 12b is a timing diagram showing interim signalsof the circuit shown in FIG. 12a . The illustrated circuit can detecttwo overlapping photon avalanches that result in a pulse that is longer(wider) than a “standard” pulse width of a single-photon pulse. Forlinearity of the measurement system, some examples identify such pulsesas being two photons rather than one photon. The comparators (Comp1 andComp2) can be single-ended- or differential-output. Various comparatortechnologies can be used, e.g., technologies that allow for propagationdelays of hundreds of picoseconds. As shown in the illustrated example,longer pulses are counted twice: once with the lower counter on Comp 1,and once with upper counter on Comp 2. The total count is then the sumof the lower counter's count and the upper counter's count. Comp2 canonly go high if the pulse is longer than one standard pulse-width,because Comp2 is only enabled once a standard pulse-width's worth oftime has passed after the onset of the pulse. This can preventdouble-counting of single photons while still providing accuratecounting of two overlapped photon pulses. Comp2 is enabled by Comp1,after a delay. The input signal is also delayed, so that it arrives atComp2 one standard pulse-width before Comp2 becomes enabled. Therefore,Comp 2's input signal is high when it becomes enabled only if the pulseis wider than one standard pulse-width. In some examples, the buffer anddelay line introduce a delay of (td−standard pulse-width). Thepropagation delay “t_(D)” is the time between when the input pulsearrives at Comp 1 and when comp 2 becomes enabled. The standardpulse-width varies by device and preamp stage. In some examples, thestandard pulse-width can be about 400 ps to about 900 ps.

The illustrated polarity in FIG. 12b are for illustration purposes onlyand is not limiting. For example, the counters can increment on anactive-low or active-high clock. Suppose a positive rising pulse isapplied to IN+ and a threshold is applied to IN−. The comparator will goLO->HI as the positive pulse crosses the threshold. This is appropriatefor an active-high counter. For a negative pulse, the inputs would beflipped in order to continue using an active high comparator. In otherimplementations there may be multiple buffers or multiplexers for signalobservation and sourcing which could invert the signal one or moretimes.

According to various embodiments, a pedestal correction factor between0.1 and 0.15 can be used. According to various embodiments, the pedestalcorrection can be determined based on the light level (e.g., high vs.low) using feedback from the adaptive threshold determining unit.Referring to FIGS. 13a and 13b , example circuit configurations ofadaptive pedestal clamping circuits, are provided. According to variousembodiments, the illustrated circuitry can stabilize the pedestal levelof short photon pulse signals. According to various embodiments, as anumber of incident photons increases, signal pedestal fluctuates due tothe high-pass filter. In short, the DC level of the signal rises abovethe original level due to high-pass filtering. However, counting photonsignals without the high-pass filter can lead to level deviations and asmaller signal, which can result in the limited dynamic range of someprior schemes. Therefore, some examples count the high-pass filter ordifferentiator output and use pedestal clamping circuitry to reducepedestal-level variation and to stabilize the inputs to the comparator.

Referring to FIGS. 13a and 13b , two embodiments of circuitconfigurations of adaptive pedestal clamping circuits are shown. In someexamples, the illustrated circuitry can stabilize the pedestal level ofshort photon pulse signals. In some examples, as a number of incidentphotons increases, a signal pedestal fluctuates due to the high-passfilter. However, counting photon signals without the high-pass filtercan lead to level deviations and a smaller signal, which can result inthe limited dynamic range of some prior schemes. Therefore, someexamples count the high-pass filter or differentiator output and usepedestal clamping circuitry to reduce pedestal-level variation and tostabilize the inputs to the comparator. In both cases of FIGS. 13a and13b , components are chosen such that VC_(ombuned_Out) is substantially1/10 TIA−V_(shaped_in), where TIA is the transimpedance amplifier. Someexamples improve the accuracy of the pedestal clamping by reducing thephase difference between the pixel-signal output and the differentiator(“HF”) output. For example, components of the circuit can be adjusted,or delay lines or other delay elements added, to keep the phase shift ofthe electronics below the pixel pulse width. Referring to FIGS. 14a-14f, graphs of pixel out and various other operations discussed above vs.time is shown. In particular, FIGS. 14a and 14b , show values of pixelout vs. time for low photon count rate and high photon count rate,respectively. FIGS. 14c and 14d , show values of differentiation out vs.time for low photon count rate and high photon count rate, respectively.FIGS. 14e and 14f , show values of adaptive pedestal clamp vs. time forlow photon count rate and high photon count rate, respectively.

With reference back to FIG. 15, a block diagram of an embodiments of theabove described blocks are provided. AS discussed above, the blockdiagram shown in FIG. 15 is designed to operate with at least one of: anavalanche process ˜100 ps, a photon pulse width <1 ns, or a quenchingtime greater than 20 ns or 50 ns. Although shown as connected on theanode side of the APDs, the measurement electronics can additionally oralternatively be connected on the cathode side. The output of thecomparator can be used by a synchronization circuit in order thequantify photon event. It should be appreciated that the PedestalControl block and the Adaptive Threshold block discussed above can beused alternatively or together.

To track photon events, one needs to do so in real-time. A real-timesystem can compute over the photons at the same rate at which theyarrive. This enables a system to continuously interpret or operate uponthe material it measures, even while constrained by finite memory.Towards this end, frontend digital circuits are shown in FIGS. 16a and16b , as alternative embodiments. The challenge FIGS. 16a and 16bresolve is that i) the digital signal arriving from the comparator isoperating with 10 ps-100 ps time-varying features owing to theavalanching events. Consequently, based on the Nyquist frequencyrequirement, a clocking frequency for capturing such changes requires aclock in the order of 10 GHz. Available electronics may not allow forsuch a fast clock. Therefore, oversample approaches are presented inFIGS. 16a and 16b enabling off the shelf products to be used. Towardsthis end, delayed versions of the comparator output (see FIG. 15) ispresented to flip flops that are responsible for clocking in the delayedcomparator signals. The frontend digitizers generate four parallel 2.5Gbps bit streams, each distributed to one of the four GTP receivers ofan FPGA, thereby achieving a target of 10 Gbps throughput needed for areal-time photon accounting. It should be noted that photon counting andphoton accounting are used interchangeably in the present disclosure.The implementation requires a tightly-controlled microwave design. Allclock and signal paths require controlled propagation delay with atolerance of about 10 ps, implying route distances should be adjustedwithin about 3 mm. In FIGS. 16a and 16b , the upstream analog circuitsof FIG. 15 have been reduced to a few elements, as shown. The inputcomparator (responsible for output of FIG. 15) converts analog photonpulse to logic signal with <100 ps transitions. Delay linesdeterministically skews the arrival of a signal so that it can beoversampled. The four clocked latches are clocked at the 2.5 GHzsampling frequency, cause stretching of the comparator output. The XORgates can set the polarity of the latch output at runtime by controllingone input. A phase lock loop (PLL) clock generator generates 156.25 MHzreference and 2.5 GHz sampling clocks. The general principle ofoperation is that the input signal into the frontend is sampled by fourseparate circuits at 2.5 GHz each, each operating slightly out of phase(i.e., 100 ps, 200 ps, 300 ps, and 400 ps). In that way, the inputsignal is actually sampled by exactly one of the four circuits every 100ps, for the 10 Gbps sampling goal. In both FIGS. 16a and 16b , XOR gatesare used as a way to synchronize the 2.5 GHz clock with clocks ofupstream circuitry, e.g., FPGA. The output can be provided directly toGTP receiver ports of the FPGA, however, if 2.5 GHz ports areunavailable, the clocked signals can be divided into parallel linesdeserializer and presented to the FPGA as parallel outputs. Therefore,if 2.5 GHz is divided into 16 deserialized lines, each line onlyrequires 156 MHz. This deserialization (two cascade versions) is shownin FIG. 16b . Therefore, the main blocks are: 1) oversampling by addingdelays to the comparator output, 2) synchronizing an otherwiseasynchronous photon digital signal to a clock having an operationfrequency F (e.g., 2.5 GHz), 3) synchronizing against downstreamcircuits, and 4) deserializing to a predetermined number of parallellines N each operating at F/N.

As yet another alternative embodiment (not shown), an applicationspecific integrated circuit can replace both frontend designs shown inFIGS. 16a and 16 b.

To graphically explain the operation, reference is made to FIG. 16awhere four latches hold the value of the pulse signal at points in time100 ps apart. While all latches are clocked simultaneously every 400 ps,the pulse signal is skewed such that it arrives at each latch exactly100 ps after it arrived at the preceding latch. The result is a 1:4deserialization. Referring to FIG. 17, a timing diagram is provided toshow the timing relationship. In FIG. 17, the latches are shown reversed(greatest skew to least skew) because the lesser skewed latchescorrespond to later points in time. At any moment, the 400 ps skewedlatch observes the comparator value from an earlier time, thus the 100ps skewed latch sees a more recent value. Suppose an incoming pulsewidth is 600 ps. Four delayed versions are shown, first one at 100 ps,second at 200 ps, third at 300 ps, and fourth at 400 ps. A clockfrequency of 2.5 GHz, translates to 400 ps period. After the first clockrising edge, all values are 0s and thus 0 is latched and clocked for allthe delayed signals. At the second clock rising edge, the 100 ps delayedversion is at 1 while the others remain at 0. Therefore, 1 is latchedand clocked for the first delayed signal and 0s are latched and clockedfor the remainder. At the third clock rising edge, all are latched andclocked at 1s. At the fourth rising edge of the clock, all are back to 0except the fourth delayed signal (the 400 ps delayed). At the fifthrising edge of the clock, the third delayed (i.e., the 300 ps delayedsignal) is changing right at the clock transition. At the last shownrising edge all delayed signals are latched at 1 except for the firstdelayed signal (i.e., the 100 ps delayed signal).

As shown in the fifth rising edge of the clock, if the latch is clockednear the input's transition, the value is indeterminate. The samplinguncertainty and jitter can be reduced using latches with low jitter andsetup/hold time. A device such as the ADCMP582 comparator withlatch-enable can fill that role.

Another alternative to the frontend digitization (not shown), includesan application specific integrated circuit (ASIC) that can clock in thecomparator output at a high frequency 10 GHz. Such a circuit requiresflip flops that can handle high frequencies such as 10 GHz. The ASICfurther includes additional blocks, e.g., counters to identify photonsand their associated arrival times.

In order to interpret information from each photon in real-time, thearrival time of every single photon is measured with some resolution,with timespan recorded or interpreted. The information contributed byeach photon is limited by time resolution, but maximized by preservingthe interpulse duration (IPD). As a result, the data rate is very highthus allowing for a real-time photon accounting. As an initial matter,it should be noted that a real-time system can compute over the photonsat the same rate at which they arrive. This capability enables a systemto continuously interpret or operate upon the material it measures, evenwhile constrained by finite memory.

Without any compression, by storing the receiver bitstream (see outputof FIGS. 16a and 16b ) as raw data in memory, photons' occurrences arerepresented spatially as a span, a sequence of ‘1’s in address-spacecorresponding to the time and duration of a photon pulse. In effect,suppose the deserialization referred to in FIG. 16b includes 64 lines(each operating at 2.5 GHz/64=39 MHz). These lines represent a frame.

Referring to FIG. 18, a block diagram is shown on how the digital engineof the photon counting operates, referred to herein as the photonpipeline. A bitstream representing a frame is presented to adiscriminator block that is configured to seek for a 0 to 1 transitionand provide a shadow timestamped data with all values 0s except wherethe transitions from 0 to 1 occur. The encoder provides a digital valueas to where the first 1 in each block is based on a priority encoder.This encoded output is then recorded as a timestamp as the timeassociated with a photon. Next, the bit which was just encoded ismasked, and the process repeated from the encoder block until allphotons in the frame are accounted for.

Referring to FIG. 19, an example discriminator circuit is shown. Thediscriminator receives an input bitstream identified as input vector.Based on its logic gates, it is configured to seek out 0-1 transitions.A 0-1 transition, represents arrival of a photon in digital values. Thephoton may remain for several is before the input bitstream goes back to0s. In the illustrated embodiment of FIG. 19, the first bit of the inputbitstream, is 0 while the second bit is a 1, representing a 0-1transition. The output of the discriminator then creates a shadowbitstream, referred to as output vector, which toggles from 0 to 1between the associated bit positions. In the input bitstream, the next 5position retain 1s, indicating the persistence of the photon pulse.Thus, in the shadow bitstream, 0s are placed in these positions. In thenext three positions of the input vector, 3 0s are provided, indicatingno photon activities. Similarly, in the shadow bitstream, the next 3position are 0s. In the input bitstream, the next two positions are isindicating a transition from a 0-1, indicative of an arrival of a newphoton. Again, in the shadow bitstream, the transition from 0-1 of theinput bitstream is represented by a 0-1 transition as well, followed bya 0. Thus for an input bitstream of 011111100011, an output vector of010000000010 is generated out of the discriminator.

Alternatively, pulse discrimination can be based on more complexcharacteristics. For example, if there are long stretches of is followedby a 0, could be related to overlapping pulses. For example, a 1followed by 5 is followed by a 0 may also cause the discriminator toprovide a 1. Referring to FIG. 20, this additional discriminationcriterion can be seen. For example, the usual condition (1) is met whenthere is a 0-1 transition. However, a second condition is met on a 1because of 5 follow-on is followed by a 0. In a third situation bothabove conditions are met.

Next, according to FIG. 21, a bitstream for a priority encoder is shownwhich provides positional values of the is in respective prioritypositions. For example, a byte having 8 bits (01000101) is passedthrough the priority encoder with the left most 1 set as priority,thereby multiple is would not cause an indeterminate output. As aresult, the output represents the value of 6 representing 1 in the6^(th) position, the left most 1. Thus, the output of the priorityencoder is 6 (i.e., 110). This positional value is recorded as thetimestamp of the photon arrival in the bitstream. Next, the bit thatprovided the output is masked and the priority encoder is rerun on00000101. This time with position 6 masked, the next position isposition 2. The priority encoder's output is then 2, and so on.

Referring to FIG. 22, exemplary operation of the priority encoding andmasking are shown for a longer bitstream. There are 21 positions (fromright to left: bit positions 0 to bit position 20). In the first roundout of the priority encoder, the 1 in position number 19 is chosen,resulting in an output of 10011. Next the bit in position 19 is maskedand the priority encoder operates on the new bitstream. The newbitstream's most significant position occupying a 1 is the position 13,resulting in an output of 01101. Next, the 1 in position 13 is masked,leaving only the 1 in position 4. Passing this new bitstream through thepriority encoder results in 00100. The result of the recordings from thepriority encoder's output as timestamps is the timing associated witheach photons that can be used to further enhance the understanding offlow cytometry.

As discussed above, the system of the present disclosure is capable ofsimultaneously providing photocurrent data as well as photon accounting.The photon accounting is described above. As for the photocurrentmeasurements, reference is made to FIG. 23. The path identified asphoton accounting is fully described above. The path identified asphotocurrent can be pursued simultaneously. Thus, when a particularGeiger mode APD encounters a photon, a differential output (D-Out)connected to the Geiger mode APD may issue a photon burst, as shown inpurple on the right of FIG. 23. When any of the Geiger mode APDsencounters photon(s), the “normal” out (N-Out) may emit a photocurrentwaveform. A low pass filter may be coupled with the normal out, asindicated in FIG. 23. Thus, a Gaussian waveform can be generated fromthe photocurrent path.

In some cases, the photon burst and the photocurrent waveform can bedetected simultaneously, or virtually simultaneously. In some examples,one or more photons (Joules) and derivative photon current (coulomb/sec)can be detected simultaneously, such as by using the example circuitillustrated in FIG. 23. Certain examples described herein make itpossible to detect all photon signal with the a relatively highsensitivity before the derivative photon current is obtained (Rayleigh,Raman, AFL).

While, the simultaneous photocurrent measurement and single-photonaccounting can be performed, and the photocurrent can generate thefamiliar Gaussian distribution as shown in FIG. 23, the single-photonaccounting pathway can also be used to generate a similar GaussianProfile. Towards this end, a moving average with a selectable windowsize can be used and overlaid over the photon timestamps. The resultingGaussian distribution is similar to the Gaussian distribution of thephotocurrent pathway of FIG. 23 when there are sufficient number ofphotons.

Generally, when measuring light, the intensity or power of the signalcorresponds to the rate of photon arrivals. In a photocurrentmeasurement regime, the rate can be measured instantaneously in the formcurrent or voltage. With discrete photon counting the rate of photonarrivals can be characterized via the average time between arrivals orequivalently, the total of photons within some time interval. With thephoton information available, it is possible to measure the number ofphotons over an arbitrary interval of time. Towards this end, a movingaverage window can be implemented. As a result the photon arrivalswithin a time interval are measured which provide an instantaneousrate=total/time. Next, the interval is shifted forward in time by somelength less than the window length. Such a moving average subtractsearlier photons from the total, and adds more recent photons, reflectinga more recent rate value. Referring to FIGS. 24 and 25, the movingaverage operation is shown graphically. In FIG. 24, each photon istimestamped in memory. As the intensity of the light source isincreased, rate of photons arrival increases. A moving average plot canprovide a Gaussian distribution similar to the photocurrent measurement.As for the moving average input, two parameters of are interest:sampling rate (fs) which is one over sampling period (Ts), whichcorrelates to the frequency at which the output is updated, and windowlength (Tw), i.e., the length of time over which the photons areconsidered. The window length must be long enough to gather sufficientphotons to make a rate measurement of sufficient accuracy. The choice ofTw is also based on signal to noise ratio and avoidance of unintendedaveraging of higher frequency components.

The moving average filter can be applied cyclically as in a circularbuffer until sufficient smoothness is achieved.

In implementing the moving average, a self-triggering scheme can beimplemented to alert of a significant likely measurement event. Forexample, if the average background photon count “Nb” over one windowlength is known, a threshold can be set at 3-sigma above the backgroundcount Nb+(3*sqrt (Nb)). When the moving window output is above thatthreshold value it indicates an extremely likely measurement event. Thatcan alert downstream analyses to begin recording photon data. Similarly,as the threshold crossing indicates the start of some measurement event,and the end of the event is when the count drops down below thethreshold again, indicating the end of the measurement event. Such atrigger can be provided to downstream analyses tools to begin and endanalysis of the photons. In order to avoid discarding photons just belowthe first threshold crossing (indicating start of an event) and justafter the second threshold crossing (indicating end of the event),photon information prior to and after the threshold crossings aremaintained in memory. For example, an additional array of circularbuffers can be used to hold the bit-stream. Each circular buffer recordsthe latest photon data each cycle. At the end of an event, one of thecircular buffers can be halted, and the data transmitted to another CPUstarting from some # of cycles prior to the original threshold crossing.In other cases the photon stream may need to be processed in real-time,rather than buffered and transferred. In that case, the stream can bedelayed by an arbitrary number of cycles using RAM or register layers,so that when the threshold crossing occurs, the processing logic isinitiated, and it is initially presented with photon data slightlybefore the threshold crossing.

Two parameters of interest from the Gaussian distribution are: Width andHeight of the Gaussian distribution. These parameters can be used toprovide a feedback signal to the light source to control the intensityin a feedback control scheme. Referring to FIG. 26, a schematic of apeak/width detection is shown utilizing a cascade of moving averagefilters (1 . . . N stages). A cascade of moving average filteradvantageously uses RAM and simple adders/subtractors rather thanprocessor resources for alternative filter schemes, such as finiteimpulse response (FIR) filters, known to a person having ordinary skillin the art. Accordingly, as shown in FIG. 26, the output of thepseudo-LPF (i.e., cascaded moving average filters) can be passed to apeak-hold circuit which will capture the maximum value following athreshold-crossing event discussed above. The half-max width can becomputed from the number of cycles until half-max is reached as thesignal declines following the peak-hold. The resolution of widthmeasurement is limited to the sample rate of the moving average, forexample 10 ns. If greater resolution is needed, a moving average withSub-clock-cycle resolution may be implemented.

As discussed above, a specialty photodiode filter is also within thescope of the present disclosure. Such a sensor is similar to the sensorarray shown in FIG. 6a or 6 b. However, instead of using quenchresistors for causing decay of signal, an active approach may be used.Such an approach replaces some or all of the quench resistors withswitches, e.g., FETs, such that when the avalanche period (on the orderof 10 ps-100 ps) has ended, the diode is quickly recharged using aswitch, rather than relying on the natural decay based on the RCconstant. Such an approach allows more photons to be arriving withoutsaturating the differentiator shown in FIG. 15. According to anotherembodiment, in addition, the front-end circuit shown in FIGS. 16a and16b (alternative approaches) or other approaches discussed herein withregards to use of an ASIC and clocking the photon signals at high rate(e.g., 10 GHz) using flip flops, can also be integrated in such asensor. According to yet another embodiment, theasynchronous-synchronous conversion of the data into timestamped photonevent and the circuitry associated therewith (see FIGS. 18-22) can alsobe integrated into the same ASIC, which will advantageously avoidcrosstalk, and other communication issues common in high-speedtransference of data. According to yet another embodiment, thesimultaneous photocurrent and photon accounting discussed herein and theassociated circuitry discussed above (including reference to FIG. 23)can also be implemented in such a sensor. Finally, and according to yetanother embodiment, data associated with the timestamped photons whichare typically held in RAM, can be placed in RAM or alternativelytransferred to non-volatile memory (e.g., FLASH) onboard the sensorASIC. Still according to yet another embodiment, feedback control of thelight source discussed above, based on photon accounting can be used tocontrol the light source and the intensity of the light source. Such anASIC allows portability and improvement of flow cytometers tremendouslyby allowing existing flow cytometers to be retrofitted with a highlycapable sensor ASIC capable of not only providing traditionalphotocurrent data, but also provide an accounting of each photon as wellas control of the light source.

Referring to FIG. 27, according to one embodiment of the presentdisclosure, a graphical representation of a system 2700 with multiplelasers is shown. FIG. 27 shows a plurality of collected signals whenmultiple lasers are used. For example, according to one embodiment,eight lasers are utilized (in the embodiment shown: 355 m, 405 nm, 488nm, 543 nm, 561 nm, 632 nm, 660 nm, and 808 nm), however, other numbersof lasers such as 2 or more are within the ambit of the presentdisclosure. While there are 8 lasers depicted in line numbers, forexample purposes, 5 sets of combinations with a maximum of 5 lasers arehighlighted, however, as discussed herein, this combination of lasers isjust one embodiment, and thus any number of lasers, in any combinationis possible. For each laser and its associated wavelength, the number ofchannels from the detectors that could be collected are identified. FIG.27 demonstrates use of multiple lasers for detection of particles in aflow stream. As described further below, by using two or more lasersparticles are detected at various positions within the flow stream.

With further reference to FIG. 27, the system 2700 includes severallaser excitations with example wavelengths described in nm and anexample detector with 42 photodetectors. While only 8 lasers are shownin the figure (355 line, 405 line, 488 line, 543 line, 561 line, 632line, 660 line, and 808 line, other laser lines are possible. In theexample shown, 42 emission lines 2701 are shown from 340.3 nm to 782.6nm, however these emission lines are based on the embodiment chosen andno such limitation is thereby intended. Below the emission lines, areshown the possible number of emission channels that can be collectedfrom any particular laser. In one example from the 355 line, 40 channels2702 are shown from 361.8 nm to 782.6 nm. The channels 2702 represents40 out of the possible 42 sensors shown in this figure. In anotherexample, from the 632 nm laser line, it is possible to collect 14emission channels 2703 are shown. Also shown would be the total numberof channels possible if a set of 5 lasers (488, 355, 405, 543, and 632)were used as shown in 2704 in which case 139 total channels areavailable. In another case for 4 lasers (488, 355, 405, 543 and 632) atotal of 99 channels would be possible as shown in 2705. Furthermore, inanother 4 laser example, (488, 355, 405, and 632) a total of 120emission channels 2706 would be available. It can be seen that differentcombinations of lasers can produce a different number of emissionchannels. Any number of lasers may be used for excitation from one tomore than one laser.

It should be appreciated that FIG. 27 describes a system wherein asingle photodetector array with a plurality of channels is used for aplurality of lasers. The plurality of channels of the photodetectorarray, are multiplexed based on time-delay (i.e., time-delaymultiplexed) in order to provide a single multiplexed signalrepresentative of all the lasers and their respective particle-generatedsignals resulting from the particle arriving in line with each of thelasers as further described in reference to FIG. 29. For example,suppose the first set of lasers shown in FIG. 27 (i.e., 488 nm, 405 nm,and 543 nm) are the three lasers used in a multi-laser system. Furthersuppose, the 488 nm is a first laser that is continuously on (see FIG.29 and its description provided below). The 488 nm laser is associatedwith 28 channels of the 42-channel photodetector array, as shown in FIG.27. When the particle in the flow stream is in line with the 488 nmlaser, for a short period of time (depending on the flow velocity, whichis a known parameter), about 28 of the 42 channels register a signal. Asdiscussed with reference to FIG. 29, apriori knowledge of flow velocityand positional relationship between the next laser can be used togenerate a trigger signal for the 405 nm laser and activate this laserin a discretized manner (see FIG. 29). At that point, for a short periodof time about 34 channels of the 42-channel photodetector array registera signal corresponding to the particle coming into view of the 405 nmlaser. Again as discussed with reference to FIG. 29, apriori knowledgeof flow velocity and positional relationship between the next laser canbe used to generate a trigger signal for the 543 nm laser and activatethis laser in a discretized manner (see FIG. 29). At that point, for ashort period of time about 23 channels of the 42-channel photodetectorarray register a signal corresponding to the particle coming into viewof the 543 nm laser. Each of these sets of channels (i.e., 28 channels,34 channels, and 23 channels) are responsible for detecting signals atdifferent time, however, these channels and their signals are thentime-delay multiplexed to generate a unified word of 85 total channelsassociated with the three lasers and their respective signals. Thistime-delay multiplexed signal from the single photodetector array isthen provided to a processor for further processing The above examplewith three lasers is provided only to demonstrate a significantadvancement in flow cytometry eliminating the need for individualphotodetector or individual photodetector arrays for each laser.However, it should be appreciated that the time-delay multiplexingdiscussed herein can be applied to any number of lasers and theirassociated particle-generated signals and the associated wavelengths.Furthermore, the time-delay multiplexed signal is provided to aprocessor accompanying a tangible non-volatile computer-readable storagemedium operably coupled to the processor such that the memory comprisesinstructions stored thereon, which when instruction are executed by theprocessor, cause the processor to disassociate the time-delaymultiplexed signal.

Referring to FIG. 28, a schematic of a multi-laser system 2800 isdepicted. Specifically, FIG. 28 depicts a system utilizing a singlefiber 2801 for delivering excitation to an optical stage 2815 as well asa single fiber 2802 for delivery of emission output from the opticalstage 2815 to a detector 2803 (while a single fiber 2801 and 2802 areshown, optionally each can be substituted with multiple fibers, each fora different wavelength).

Furthermore, FIG. 28 shows an optical path of a photon spectral flowcytometer. Collimated multiple laser beams are combined by a beamcombiner and beam power is monitored and a feedback signal generated viaindividual Photo Diodes (PD) 2804 monitoring each laser output as shownin FIG. 28. It should be appreciated that the PD 2804 can be replaced byany detector selected from the group consisting of photomultiplier tubes(PMT), Geiger avalanche photodiode (APD), silicon photomultipliers(SiPM), and combinations thereof, as is well known by a person havingordinary skill in the art. Combined laser beams are aligned, and top-hatbeam shaping can be applied before a focus lens (CYL-L) 2805 is appliedto an interrogation point 2806 in the flow channel. The interrogationpoint 2806 is a focus point that is in about the center of the flowchannel. Focused laser spot can be modulated to excite particlessequentially. Emitted fluorescent light is collected by spherical mirror2807 and directed to the fiber 2802 where individual laser emissionlines are separated by wavelength via a polarization maintaining (PM)fiber and sent to a spectral sensor unit 2803 which can cover the fullwavelength range, for example a 42CH photon sensor array. It is possibleto use a fiber channel (or ferrule connector), known as an FC connector2812 for the connection of the optical fiber which allows the system tobe alignment free. Of particular note are key features of the examplemulti-laser system 2800 which include plug-and-play laser function dueto inclusion of fiber collimator 2808 via for example FC connectors,although other connector types could be substituted. In addition,top-hat laser beam shaping can be achieved via the aspherical mirror2807 as shown in FIG. 28. In addition, because of the use ofcatadioptric optics 2809 shown in FIG. 28, a wide wavelength range ispossible. In addition, higher fluorescence collection efficiency can beobtained by spherical mirror and aspheric lens of the catadioptricconfiguration as shown in FIG. 28. By separating the laser emissionlines, (see FIG. 29) full spectral detection by the use of time delaycan be accomplished using a single spectral sensor or multiple spectralsensors. The use of optical fibers minimizes stray laser light and addan aspect of laser safety. By use of individual optical fibers for laserdelivery via FC connectors, to an optical unit 2810, each laser line canbe transmitted to the flow chamber by use of dichroic mirrors 2820 (DM1,DM2, etc.) which transmit only the laser lines but allow laser powermonitoring via the reflected signal that is transmitted to eachindividual PD 2804. This feature allows power manipulation of each ofthe lasers. The multi-laser system 2800 shown in FIG. 28 also shows theuse of an X-Y shifter 2821 to make minor adjustments to the laser beamsexiting on the single fiber 2801. In some embodiments, light detectionincludes the photodetector 2804 and an optical adjustment component 3001(see FIG. 30) that is configured to reduce the amount of light that isconveyed from a 1^(st) laser to the photodetector 2804. In someembodiments, light from the laser is detected by the photodetector 2804directly. In other embodiments, light from the laser is detected by thephotodetector 2804 through a dichroic mirror 2820. In some embodimentsthe interior of the optical unit 2810 can be covered with a blackeningmetamaterial 2822 to reduce reflection, as is well known to a personhaving ordinary skill in the art.

Referring to FIG. 29, a timing chart 2900 is shown depicting theoperational timing of the lasers discussed in reference to FIGS. 27 and28. FIG. 29 shows the use of sequential lasers for a flow cytometryinstrument. The ordinate shows lasers with a 1^(st) laser 2901 ₁ to a5^(th) laser 2901 ₅ (i.e., the 1^(st) laser 2901 ₁, a 2^(nd) laser 2901₂, a 3^(rd) laser 2901 ₃, a 4^(th) laser 2901 ₄, and the 5^(th) laser2901 ₅), shown as an example embodiment, with the 1^(st) laser 2901 ₁ asalways on (i.e., continuously irradiating). The 1^(st) laser 2901 ₁ canbe any wavelength, such as 488 nm shown, or 808 nm or other wavelengthas desired. As a particle 2903 (shown as n^(th) particle) travelsthrough a fluidic pathway 2904 (e.g., a flow path of the flow cytometryinstrument), it first passes by the 1^(st) laser 2901 ₁, resulting in atrigger signal 2905 for the follow-on lasers. Since the velocity ofparticle flow is known and the distance between the 1^(st) laser 2901 ₁and, e.g., the 2^(nd) laser 2901 ₂ is also known, when the particlereaches the 2^(nd) laser 2901 ₂, the trigger signal 2905 from the 1^(st)laser 2901 ₁ is translated to a trigger signal 29062 for the 2^(nd)laser 2901 ₁, and thus the 2^(nd) laser 2901 ₂ is activated for adiscrete interval as shown in FIG. 29. The particle then proceeds to the3^(rd) laser 2901 ₃, and so on. At this point, the 2^(nd) laser 2901 ₂has been turned off, and the 3^(rd) laser 2901 ₃ is activated, againbased on apriori knowledge of flow velocity and distance between the1^(st) laser 2901 ₁ and the 3^(rd) laser 2901 ₃. At this point thetrigger signal 2905 from the 1^(st) laser 2901 ₁ is translated to atrigger signal 2906 ₃ for the 3^(rd) laser 2901 ₃, and thus the 3^(rd)laser 2901 ₃ is activated for a discrete interval as shown in FIG. 29.The particle then proceeds to the 4^(th) laser 2901 ₄. At this point,the 2^(nd) laser 2901 ₂ and the 3^(rd) laser 2901 ₃ have been turnedoff, and the 4^(th) laser 2901 ₄ is activated, again based on aprioriknowledge of flow velocity and distance between the 1^(st) laser 2901 ₁and the 4^(th) laser 2901 ₄. At this point the trigger signal 2905 fromthe 1^(st) laser 2901 ₁ is translated to a trigger signal 2906 ₄ for the4^(th) laser, and thus the 4th laser 2901 ₄ is activated for a discreteinterval as shown in FIG. 29. Finally, the particle proceeds to the5^(th) laser 2901 ₅. At this point, the 2^(nd) laser 2901 ₂, the 3^(rd)laser 2901 ₃, and the 4^(th) laser 2901 ₄ have been turned off, and the5^(th) laser 2901 ₅ is activated, again based on apriori knowledge offlow velocity and distance between the 1^(st) laser 2901 ₁ and the5^(th) laser 2901 ₅. At this point the trigger signal 2905 from the1^(st) laser is translated to a trigger signal 2906 ₅ for the 5^(th)laser, and thus the 5^(th) laser 2901 ₅ is activated for a discreteinterval as shown in FIG. 29. It should be appreciated that the 1^(st)laser 2901 ₁ which is continuously on has a wavelength that is shorterthan the other lasers (e.g., the 1^(st) laser 2901 ₁ is shown to be 488nm, however, the 3^(rd) laser 2901 ₃ and the 5^(th) laser 2901 ₅ havewavelengths that are longer); or alternatively, the 1^(st) laser 2901 ₁may have a wavelength that is longer than the other lasers, e.g., the2^(nd) laser 2901 ₂ and the 4^(th) laser 2901 ₄. As discussed above, itshould further be appreciated that the trigger signal 2905 from the1^(st) laser 2901 ₁ is translated to trigger signals of the other lasersfor their respective activations in a discrete manner. For example, asshown in FIG. 29, the 2^(nd) laser 2901 ₂ is only activated for a shortperiod in response to the trigger signal 2905 of the 1^(st) laser. Itshould also be appreciated that, and as described above, each of thelasers (i.e., the 2^(nd) laser 2901 ₂, the 3^(rd) laser 2901 ₃, the4^(th) laser 2901 ₄, and the 5^(th) laser 2901 ₅) are independentlytriggered by the 1^(st) laser 2901 ₁ based on the flow velocity andapriori knowledge of the distance between the 1^(st) laser 2901 ₁ andthe other lasers. It should further be appreciated that the downstreamlasers (i.e., the 2^(nd) laser 2901 ₂, the 3^(rd) laser 2901 ₃, the4^(th) laser 2901 ₄, and the 5^(th) laser 2901 ₅) are activated anddeactivated so there is no overlap of activation between these lasers.For example, as shown in the non-limiting example of FIG. 29 thedistance between each subsequent laser and the 1^(st) laser 2901 ₁ is amultiple of 50 μm, resulting in Δt multiple of 25 μs for a flow velocityof 2 m/s (i.e., 50 μm/2 m/s) which results in the aforementioned 25 μsmultiples, as provided in the example box 2908. Therefore, the triggersignal 29062 for the 2^(nd) laser 2901 ₂ and the trigger signal 2906 ₃for the 3^(rd) laser 2901 ₃ are at least 25 μs apart, necessitating apulse width of discrete activation shorter than 25 μs. For example, thetrigger signal 29062 of the 2^(nd) laser 2901 ₂ is 25 μs away from thetrigger signal 2906 ₃ of the 3^(rd) laser 2901 ₃, however, the 2^(nd)laser 2901 ₂ necessitates a pulse width of less than 25 μs. It should beappreciated that these are only example values, and no limitations areintended by these numbers. In other words, the flow velocity can befaster or slower; or the space between the subsequent lasers can beshorter or longer from the 1^(st) laser 2901 ₁, all of which affect theaforementioned multiples of Δt and consequently the pulse width of eachdiscretized activation of the subsequent lasers. It should also beappreciated that the triggering of the subsequent lasers is based on theaforementioned calculations performed by a processor, e.g., theprocessor system 104 shown in FIG. 1 or the processor 486 shown in FIG.4, based on execution of computer program instructions configured tooperate measurement systems or capture measurements as describedhereabove. Processor 486 can send messages and receive data, includingprogram code, through network 450, network link 416, and communicationinterface 415. For example, a server can store requested code for anapplication program (e.g., a JAVA applet) on a tangible non-volatilecomputer-readable storage medium to which it is connected. The servercan retrieve the code from the medium and transmit it through network450 to communication interface 415. The received code can be executed byprocessor 486 as it is received, or stored in data storage system 440for later execution.

As described herein, the term “continuous” is used herein in itsconventional sense continuous wave (CW) to refer to laser irradiation ofthe flow stream which is constant and not otherwise interrupted for aduration that a sample of interest is flowed through the flow stream. Insome embodiments, a laser configured for continuous irradiation is alaser that is unobscured (i.e., not intermittently blocked with anoptical modulator 3001 (see FIG. 30)). In certain instances, continuousirradiation of the flow stream with the laser includes maintaining aconstant laser irradiation intensity. In certain embodiments, the laserconfigured for continuous irradiation of the flow stream exhibits nochange in intensity for the duration that a sample is flowed through theflow stream. The intensity of light output by the laser configured forcontinuous irradiation can be measured with any convenient protocol,including but not limited to, a photodetector 2804 (see FIG. 28) orother types of photodetectors.

The term “discrete interval” is used herein in its conventional sense torefer to irradiation of the flow stream for a predetermined duration oftime followed by a period of time where the flow stream is notirradiated by the laser (e.g., by turning off the laser or by modulatingthe laser such as with an optical modulator 3001 (see FIG. 30)).

Any convenient protocol can be used to provide intermittent irradiation,such as an electronic switch for turning the laser on-and-off, such as aswitch that is computer-controlled and triggered based on a data signal(e.g., received, or inputted data signal) as described in detail above.In some embodiments, lasers are configured for irradiation in discreteintervals by intermittently exposing the laser beam of each laser to anoptical modulator 3001 (see FIG. 30).

In some embodiments, methods include continuously irradiation a flowstream with a first laser and irradiating the flow stream in discreteintervals with a plurality of lasers, such as with 2 or more lasers,such as 3 or more lasers and including with 4 or more lasers. In certaininstances, the flow stream is irradiated with the plurality of lasers atpositions downstream from irradiation of the flow stream by the firstlaser. In some instances, the stream is irradiated with the plurality oflasers at positions that are spaced apart from each other. In someinstances, the stream is irradiated with each of the plurality of lasersfor discrete intervals well known to a person having ordinary skill inthe art. The irradiation interval is determined by the velocity of thestream as shown in FIG. 29. In other embodiments, each signal receivedfrom a cell, excited by a laser, for example by the 1^(st) laser, willtravel through 2802 to the spectral detection system 2803 and be timestamped 2601 and stored in a computer. When that same cell moves throughthe flowing stream and is excited by laser 2, the received signal willalso pass through 2802 to the spectral detection system 2803 and be timestamped 2601 and stored in a delay buffer 2602 in a processor or amemory associated with a processor. This process is repeated for allavailable lasers, and finally all of the signals from a single cell willbe recorded on the computer system. Using this method, a single detectorarray can be used to collect data from each cell as it flow past alllasers. This time delay mechanism allows a single detector array to beused for all signal collection, instead of using multiple detectors foreach laser as is the current practice in flow cytometry. Aspects of thepresent disclosure include systems for irradiating particles in a flowstream. Systems according to certain embodiments include a first laserconfigured for continuous irradiation of a flow stream and a secondlaser configured for irradiation of the flow stream in discreteintervals where each discrete interval of irradiation by the secondlaser is triggered by irradiation of a particle in the flow stream withthe first laser. In some embodiments, the 2^(nd), 3^(rd), 4^(th), orn^(th) lasers may be modulated by use for example an optical modulator3001 (See FIG. 30) whereby the laser irradiation of the flow stream iscontrolled by the modulation frequency. Such pulse rates are well knownbased on the particular excited molecule of interest such as typicalwell know flow cytometry fluorochromes. Such modulation frequencies arewell known to a person having ordinary skill in the art.

Typical stream velocities are well known to a person having ordinaryskill in the art.

In some embodiments, the methods of the present disclosure includeactivating one or more downstream lasers (i.e., the 2^(nd) laser 2901 ₂,the 3^(rd) laser 2901 ₃, the 4^(th) laser 2901 ₄, and the 5^(th) laser2901 ₅), in response to irradiation of a particle in the flow stream bythe 1st laser 2901 i. In some embodiments, methods include directinglight from one or more of the downstream lasers with a beam diverter tothe flow stream in response to irradiation of a particle by the 1^(st)laser 2901 ₁ as shown in FIG. 30, which is a block diagram of an opticalsystem 3000. In some instances, the beam diverter is an optical-opticaldevice 3001 such as an optical deflector (AOD) or an acousto-opticalmodulator (AOM). In some instances, the beam diverter is anelectro-optical device such as an electro-optical deflector (EOD) or anelectro-optical modulator (EOM) or an acoustic optical modulator.

The block diagram shown in FIG. 30 depicts a flow particle signalsimulator by laser and optical modulator without any fluidics. Thissimulator provides precise analysis of detected signals. By use of theAOM 3001 it is possible to modulate the laser intensity and deflect beamby use of carrier frequency modulation. By use of a modulating laserbeam, it is possible to pulse the beam at any useful rate, for example,for a modulation pulse at a rate to collect single photon pulses asshown in FIG. 30. Such pulse rates are well known based on theparticular excited molecule of interest as known to a person havingordinary skill in the art. The simulator as shown in FIG. 30 can thencompare between the photo electron (PE) number and the photocurrentintensity and phase difference. This comparison allows the definition ofphoton statistics and the theoretical coefficient of variation (CV) ofdetected signals.

Laser modulation for sequential excitation allows for low crossoversignals and reduces power consumption as well as unwanted heat output.By ensuring lasers are connected via optical fibers, no external lightis present reducing both signal loss and laser exposure. Currently usedfree space optics for multiple wavelengths is difficult to alignprecisely and spot position is sensitive to stress and vibration.Referring to FIG. 31 a schematic of component integration for opticalsystems discussed herein is provided. Fiber coupling from laser diodeusing FC connectors 3101 or similar type connectors enablesplug-and-play function of laser excitation. Sequential excitation asthose shown in the timing chart 2900 also reduces heat load on thesystem which provides a significant advantage, allowing multiple laserdiode units to be integrated into a small box 3110 facilitating ease ofcontrol of high-speed laser modulation as shown in FIG. 31. In someembodiments, the control for laser modulation may be contained within amodule similar to 3110 which contains a power supply 3111, a digitalcontrol interface 3112 or an alternative modulation interface, as wellas coaxial connectors 3113 which may contain a primary modulationinterface, a low voltage threshold level control and may be coupled via50 ohm DC-coupling or similar. In addition, using direct laser beamsthere is a significant challenge with pointing stability, however, usingfiber optics with FC or similar connectors provides for a significantstable beam location and therefore reduces alignment difficulties. Themodule for management of laser modulation is shown as 3110.

Those having ordinary skill in the art will recognize that numerousmodifications can be made to the specific implementations describedabove. The implementations should not be limited to the particularlimitations described. Other implementations may be possible.

1. A flow cytometry system comprising: a first laser configured forcontinuous irradiation of a flow stream; one or more second lasersconfigured for irradiation of the flow stream in discrete intervals,wherein each discrete interval of irradiation by the one or more secondlasers is triggered by irradiation of one or more particles in the flowstream with the first laser; a light detection system comprising: asingle fiber optic configured to collect light from the one or moreparticles in the flow stream irradiated by each of the first and each ofthe one or more second lasers; and a single photodetector configured todetect light conveyed by the single fiber optic; and a processoraccompanying a tangible non-volatile computer-readable storage mediumoperably coupled to the processor wherein the memory comprisesinstructions stored thereon, which when instruction are executed by theprocessor, cause the processor to calculate timing associated with eachdiscrete interval of irradiation of the flow stream by each of the oneor more second lasers.
 2. The system of claim 1, wherein each of the oneor more second lasers is configured to irradiate the flow stream at aposition downstream from the first laser.
 3. The system of claim 1,wherein the wavelength of light from the first laser is shorter thanlight from one or more of each of the one or more second lasers.
 4. Thesystem of claim 1, wherein the wavelength of light from the first laseris longer than wavelength of light from one or more of each of the oneor more second lasers.
 5. The system of claim 2, wherein each of the oneor more second lasers outputs a different wavelength of light.
 6. Thesystem of claim 2, wherein each discrete interval of irradiation of theflow stream by each of the one or more second lasers is independentlytriggered by irradiation of the one or more particles in the flow streamwith the first laser.
 7. The system of claim 1, wherein the memorycomprises instructions stored thereon, which when the instructions areexecuted by the processor, cause the processor to calculate timing ofirradiation of the flow stream by each of the one or more second lasersby: irradiating the one or more particles in the flow stream with thefirst laser; detecting light from the flow stream in response toirradiation of the one or more particles with the first laser; andcalculating a time interval between irradiation of the one or moreparticles by the first laser and each of the one or more second lasers.8. The system of claim 1, wherein the system further comprises amodulator configured to modulate each of the one or more second lasers.9. The system of claim 8, wherein the memory of the processor comprisesinstructions stored thereon, which when instructions are executed by theprocessor, cause the processor to modulate the one or more second lasersin response to irradiation of the one or more particles by the firstlaser.
 10. The system of claim 1, wherein the system further comprises amodulation device positioned in the beam path between the one or moresecond lasers and the flow stream, wherein the modulation device isconfigured to divert light from the one or more second lasers away fromthe flow stream.
 11. The system of claim 10, wherein the beam diverteris configured to direct light from the one or more second lasers to theflow stream in response to irradiation of the one or more particles bythe first laser.
 12. The system of claim 11, wherein the modulationdevice comprises an acousto-optical device.
 13. The system of claim 1,further comprising a light detection system, wherein the light detectionsystem comprises: a single photodetector; and an optical adjustmentcomponent configured to modulate an amount of light that is conveyedfrom the first laser to the photodetector.
 14. The system of claim 13,wherein the optical adjustment component is a dichroic filter.
 15. Thesystem of claim 1, wherein the single photodetector is a photodetectorarray.
 16. The system of claim 1, wherein the single photodetector isselected from the group consisting of photomultiplier tubes (PMT),Geiger avalanche photodiode (APD), silicon photomultipliers (SiPM), andcombinations thereof.
 17. A flow cytometry system comprising: a firstlaser configured for continuous irradiation of a flow stream; and asecond laser configured for irradiation of the flow stream in discreteintervals, wherein each discrete interval of irradiation by the secondlaser is triggered by irradiation of one or more particles in the flowstream with the first laser.
 18. A flow cytometry system comprising: afirst laser configured for continuous irradiation of a flow stream; oneor more second lasers configured for irradiation of the flow stream indiscrete intervals, wherein each discrete interval of irradiation by theone or more second lasers is triggered by irradiation of one or moreparticles in the flow stream with the first laser; and a light detectionsystem comprising: a single fiber optic configured to collect light fromone or more particles in the flow stream irradiated by each of the firstand each of the one or more second lasers; and a single photodetectorarray having a plurality of channels configured to detect light conveyedby the single fiber optic, wherein one or more channels of the pluralityof channels of the single photodetector array is associated with acorresponding wavelength of the first laser and each of the one or moresecond lasers such that signals from the one or more channels of theplurality of channels are time-delay multiplexed.
 19. The system ofclaim 18, wherein the single photodetector array has 42 channels. 20.The system of claim 18, wherein the time-delay multiplexed signal isprovided to a processor accompanying a tangible non-volatilecomputer-readable storage medium operably coupled to the processorwherein the memory comprises instructions stored thereon, which wheninstruction are executed by the processor, cause the processor todisassociate the time-delay multiplexed signal.